Gene Expression in Trypanosomatid Parasites

The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa

Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene

Sequence comparisons of U2 snRNA genes and flanking regions from T. cruzi (CL Brener Non-Esmeraldo-like). Sequences from the genes located on chromosomes 23, 37 and 6 are shown. The U2 snRNA gene from chromosome 23 is presented in blue font. The position of boxes A and B is indicated. Sequence numbers are relative to the TSS (+1) from the U2 snRNA. (PDF 1404 kb

Gene organization and sequence analyses of transfer RNA genes in Trypanosomatid parasites

<p>Abstract</p> <p>Background</p> <p>The protozoan pathogens <it>Leishmania major</it>, <it>Trypanosoma brucei </it>and <it>Trypanosoma cruzi </it>(the Tritryps) are parasites that produce devastating human diseases. These organisms show very unusual mechanisms of gene expression, such as polycistronic transcription. We are interested in the study of tRNA genes, which are transcribed by RNA polymerase III (Pol III). To analyze the sequences and genomic organization of tRNA genes and other Pol III-transcribed genes, we have performed an <it>in silico </it>analysis of the Tritryps genome sequences.</p> <p>Results</p> <p>Our analysis indicated the presence of 83, 66 and 120 genes in <it>L. major, T. brucei </it>and <it>T. cruzi</it>, respectively. These numbers include several previously unannotated selenocysteine (Sec) tRNA genes. Most tRNA genes are organized into clusters of 2 to 10 genes that may contain other Pol III-transcribed genes. The distribution of genes in the <it>L. major </it>genome does not seem to be totally random, like in most organisms. While the majority of the tRNA clusters do not show synteny (conservation of gene order) between the Tritryps, a cluster of 13 Pol III genes that is highly syntenic was identified. We have determined consensus sequences for the putative promoter regions (Boxes A and B) of the Tritryps tRNA genes, and specific changes were found in tRNA-Sec genes. Analysis of transcription termination signals of the tRNAs (clusters of Ts) showed differences between <it>T. cruzi </it>and the other two species. We have also identified several tRNA isodecoder genes (having the same anticodon, but different sequences elsewhere in the tRNA body) in the Tritryps.</p> <p>Conclusion</p> <p>A low number of tRNA genes is present in Tritryps. The overall weak synteny that they show indicates a reduced importance of genome location of Pol III genes compared to protein-coding genes. The fact that some of the differences between isodecoder genes occur in the internal promoter elements suggests that differential control of the expression of some isoacceptor tRNA genes in Tritryps is possible. The special characteristics found in Boxes A and B from tRNA-Sec genes from Tritryps indicate that the mechanisms that regulate their transcription might be different from those of other tRNA genes.</p

Comparación de métodos para analizar materia orgánica en materiales orgánicos: Comparison of methods for analyzing organic matter in organic materials

Se compararon dos métodos (combustión húmeda y calcinación a 550 ºC) para la determinación de la materia orgánica en materiales orgánicos. En el ensayo participaron nueve laboratorios, empleando cinco materiales orgánicos (tres composts y dos sustratos), analizados por cuadruplicado. La precisión se evaluó calculando la repetibilidad y la reproducibilidad, según la norma COVENIN 2972-92 (ISO 5725-86). No se encontraron diferencias significativas entre métodos, laboratorios, ni réplicas, por lo que se asume que los métodos comparados generan resultados estadísticamente iguales. La precisión evaluada a través de la repetibilidad y la reproducibilidad no resultó dependiente del nivel de materia orgánica. Los menores valores de repetibilidad y reproducibilidad obtenidos con el método de calcinación indican que esta modalidad analítica es más precisa. Se recomienda la aplicación del método de calcinación porque se basa en un procedimiento rápido, sencillo, no consume reactivos químicos y no genera desechos tóxicos para el ambiente y la salud

VIII Encuentro de Docentes e Investigadores en Historia del Diseño, la Arquitectura y la Ciudad

Acta de congresoLa conmemoración de los cien años de la Reforma Universitaria de 1918 se presentó como una ocasión propicia para debatir el rol de la historia, la teoría y la crítica en la formación y en la práctica profesional de diseñadores, arquitectos y urbanistas. En ese marco el VIII Encuentro de Docentes e Investigadores en Historia del Diseño, la Arquitectura y la Ciudad constituyó un espacio de intercambio y reflexión cuya realización ha sido posible gracias a la colaboración entre Facultades de Arquitectura, Urbanismo y Diseño de la Universidad Nacional y la Facultad de Arquitectura de la Universidad Católica de Córdoba, contando además con la activa participación de mayoría de las Facultades, Centros e Institutos de Historia de la Arquitectura del país y la región. Orientado en su convocatoria tanto a docentes como a estudiantes de Arquitectura y Diseño Industrial de todos los niveles de la FAUD-UNC promovió el debate de ideas a partir de experiencias concretas en instancias tales como mesas temáticas de carácter interdisciplinario, que adoptaron la modalidad de presentación de ponencias, entre otras actividades. En el ámbito de VIII Encuentro, desarrollado en la sede Ciudad Universitaria de Córdoba, se desplegaron numerosas posiciones sobre la enseñanza, la investigación y la formación en historia, teoría y crítica del diseño, la arquitectura y la ciudad; sumándose el aporte realizado a través de sus respectivas conferencias de Ana Clarisa Agüero, Bibiana Cicutti, Fernando Aliata y Alberto Petrina. El conjunto de ponencias que se publican en este Repositorio de la UNC son el resultado de dos intensas jornadas de exposiciones, cuyos contenidos han posibilitado actualizar viejos dilemas y promover nuevos debates. El evento recibió el apoyo de las autoridades de la FAUD-UNC, en especial de la Secretaría de Investigación y de la Biblioteca de nuestra casa, como así también de la Facultad de Arquitectura de la UCC; va para todos ellos un especial agradecimiento

Nucleolar Structure and Function in Trypanosomatid Protozoa

The nucleolus is the conspicuous nuclear body where ribosomal RNA genes are transcribed by RNA polymerase I, pre-ribosomal RNA is processed, and ribosomal subunits are assembled. Other important functions have been attributed to the nucleolus over the years. Here we review the current knowledge about the structure and function of the nucleolus in the trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp., which represent one of the earliest branching lineages among the eukaryotes. These protozoan parasites present a single nucleolus that is preserved throughout the closed nuclear division, and that seems to lack fibrillar centers. Trypanosomatids possess a relatively low number of rRNA genes, which encode rRNA molecules that contain large expansion segments, including several that are trypanosomatid-specific. Notably, the large subunit rRNA (28S-type) is fragmented into two large and four small rRNA species. Hence, compared to other organisms, the rRNA primary transcript requires additional processing steps in trypanosomatids. Accordingly, this group of parasites contains the highest number ever reported of snoRNAs that participate in rRNA processing. The number of modified rRNA nucleotides in trypanosomatids is also higher than in other organisms. Regarding the structure and biogenesis of the ribosomes, recent cryo-electron microscopy analyses have revealed several trypanosomatid-specific features that are discussed here. Additional functions of the nucleolus in trypanosomatids are also reviewed

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major

In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1

TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major